Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3–10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3′3′-cyclic GMP–AMP define a mechanism of metal-independent hydrolysis 3′ of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation 1,2 . Phages use several strategies to defeat host CRISPR and restriction-modification systems [3][4][5][6][7][8][9][10] , but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3′3′-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3′ of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.
capable of degrading cCMP ( Fig. 2d and Extended Data Fig. 5a). We used structure prediction to analyse each protein encoded in these regions and identified that the uncharacterized SBSphiJ gene 147 encodes a protein with predicted homology to known metallo β-lactamase (MBL) fold RNase and phosphodiesterase enzymes ( Fig. 2d and Extended Data Fig. 5a, b). Recombinant protein produced from gene 147 rapidly degrades the Pycsar signals cCMP and cUMP (Fig. 2e, f and Extended Data Fig. 5c-f), and we named this anti-Pycsar protein Apyc1 (European Nucleotide Archive genome accession number ERS1981056). SBSphiJ Apyc1 efficiently hydrolyses a wide range of cyclic mononucleotides (Fig. 2f), exhibiting an atypically relaxed nucleobase specificity that enables targeting of cyclic pyrimidine signals used in Pycsar immunity.
Immune evasion genes frequently cluster together in the genomes of phages to form anti-defence islands 7,14 . Consistent with a role in CBASS evasion, T4 Acb1 is encoded adjacent to internal protein I (ipI), a phage inhibitor required to evade the E. coli restriction enzyme gmrS/gmrD that recognizes glucosylated cytosine bases present in T4 genomic DNA (ref. 15 ; Fig. 2g). Apyc1 is the first identified anti-defence gene in SBSphiJ, limiting comparative analysis with other genes in this phage. However, Apyc1 is encoded adjacent to a series of small proteins of unknown function, suggesting that this variable locus in SBSphiJ-family phages may contribute to evasion of other antiphage defence systems (Fig. 2h). To discover further Acb and Apyc proteins, we searched for proteins related to Acb1 and Apyc1 within phage genomes and prophage sequences (Fig. 2i, j). Analysis of T4 Acb1 identified 281 related protein sequences with about 97% predicted to be of phage origin. We cloned and tested a further 9 acb1 genes and observed that each recombinant Acb1 protein efficiently cleaved the CBASS signals 3′3′-cGAMP and cAAA ( Fig. 2i and Extended Data Fig. 6a). We identified 107 proteins related to Apyc1 present in phage genomes (Fig. 2j) and also found many closely related bacterial proteins encoded in diverse bacterial orders (Extended Data Fig. 6b). Similar to SBSphiJ Apyc1, closely related phage and bacterial Apyc1-like proteins cleaved cyclic mononucleotides with broad specificity ( Fig. 2j and Extended Data Fig. 6c). By contrast, the closely related B. subtilis enzymes YhfI (GenBank accession number NP_388905.1) and MBL phosphodiesterase (GenBank accession number WP_013351727.1) exhibited a strong preference for cAMP/cGMP over cCMP/cUMP cleavage, confirming that relaxed nucleotide specificity and Pycsar signal degradation are unique to Apyc1 and not general features of MBL phosphodiesterase enzymes (Extended Data Fig. 6d). The observation of Apyc1 homologues encoded in bacteria may be explained by the presence of cryptic prophages present in bacterial genomes, but also raises the intriguing possibility that host Apyc1 enzymes may play a role in regulating Pycsar defence or other cNMP-based signalling systems. In total, our analysis identified 273 Acb1 and 107 Apyc1 phage proteins, demonstrating that cyclic nucleotide-degrading enzymes constitute a widespread form of anti-CBASS and anti-Pycsar evasion.

Mechanisms of cyclic nucleotide cleavage
We next determined crystal structures of Acb1 to define the mechanism of anti-CBASS evasion. Structures of Acb1 from the Erwinia phage FBB1 in the apo state (1.1 Å) and in complex with 3′3′-cGAMP (1.2 Å) reveal that Acb1 adopts a compact 2H phosphoesterase fold with six central β-strands that form a U-shaped ligand-binding pocket (Fig. 3a, Extended Data Fig. 7a and Supplementary Table 2). On substrate recognition, the flexible carboxy-terminal residues 145-152 form an ordered lid that closes over the top of the captured 3′3′-cGAMP ligand ( Fig. 3a and Extended Data Fig. 7b). Acb1 ligand recognition is primarily independent of base identity, with the conserved aromatic residues Y12, W74, F107 and W147 forming stacking interactions with the face of each nucleobase (Fig. 3b). However, base-specific contact occurs between E141 and the 3′3′-cGAMP adenosine N6 position, explaining why at least one adenosine is required for cleavage ( Fig. 2c and Extended Data Fig. 7c). Although overall lack of sequence-specific contacts allows Acb1 to target a broad range of CBASS cyclic nucleotide signals, the Acb1 binding pocket can accommodate only cyclic dinucleotide or trinucleotide species. Structural clashes prevent recognition of larger cyclic oligonucleotides with >3 bases, and we confirmed that Acb1 is unable to degrade cyclic tetra-adenylate (cA 4 ) rings common in type III clustered regularly interspaced short palindromic repeats (CRISPR) immunity 16,17 (Extended Data Fig. 7d). Acb1-nucleotide interactions contort 3′3′-cGAMP into a highly strained conformation in which the adenosine base is rotated about 65° relative to the in-solution or receptor-bound conformation, repositioning the 2′ OH for attack on the 3′-5′ bond 18,19 (Fig. 3c). In the Acb1-3′3′-cGAMP structure, the scissile phosphate is positioned over an active-site HxT/HxT tetrad (H44, T46, H113, T115) for acid-base catalysis and the ligand is fully hydrolysed into the linear product G[3′-5′]pAp[3′] (GpAp) (Fig. 3d and Extended Data Fig. 7e). We tracked cleavage reactions in vitro using high-performance liquid chromatography (HPLC) and confirmed that Acb1 cleaves 3′ of adenosine residues in a two-step, metal-independent reaction that proceeds through a cyclic a c  Fig. 7f). Substitutions of conserved active-site and nucleotide-coordinating residues disrupt enzyme function and highlight the critical role for contacts stabilizing the rotated adenine base in Acb1 cyclic nucleotide cleavage (Fig. 3e).
To compare mechanisms of anti-CBASS and anti-Pycsar evasion, we determined the crystal structure of Apyc1 from the phage Bsp38 (2.7 Å) as well as structures of Paenibacillus Apyc1 proteins (1.5 Å and 1.8 Å). These structures confirm that Apyc1 is a member of the class II phosphodiesterase enzymes, which exhibit an MBL fold and have no structural or mechanistic homology to Acb1 (ref. 20 Table 2). Similar to other structurally characterized class II phosphodiesterases such as B. subtilis YhfI, yeast Saccharomyces cerevisiae PDE1 or widely distributed RNase Z proteins 21,22 , Apyc1 is a homodimer with a highly conserved HxHxDH motif that coordinates two Zn 2+ ions that bind phosphate groups to position cyclic nucleotides for cleavage (Extended Data Fig. 8a-c). In a structure of Paenibacillus Apyc1 co-crystallized in the presence of nonhydrolysable cAMP, we observed strong electron density near the Zn 2+ ions and more diffuse density in the nucleobase pocket, consistent with specific coordination of the phosphate and ribose backbone of cyclic mononucleotides and weaker nucleobase specificity within the enzyme active site (Extended Data Fig. 8d). Structural comparison of Apyc1 and B. subtilis YhfI also reveals that Apyc1 enzymes contain an extended loop that reaches into the nucleotide-binding pocket, potentially enabling stable binding of smaller cyclic pyrimidine substrates (Extended Data Fig. 8b). We confirmed the critical role for Apyc1 metal-coordinating residues and identified E74 and Y112 from the opposing protomer as further catalytic residues required for cCMP hydrolysis and release of the reaction product 5′-CMP (Extended Data Fig. 8e, f). Together, these findings demonstrate that Acb1 and Apyc1 constitute separate families of immune evasion proteins and explain the distinct reaction mechanisms that degrade CBASS or Pycsar cyclic nucleotide signals (Fig. 3f).

Acb1 and Apyc1 subvert host immunity
CBASS and Pycsar antiphage defence requires cyclic nucleotidedependent activation of downstream effector proteins that induce cell death 1,2,23-26 . Using a panel of CBASS nuclease and phospholipase effectors from Vibrio cholerae, Enterobacter cloacae and Burkholderia pseudomallei, we reconstituted CBASS signalling in vitro and observed that Acb1 potently inhibited activation of both cyclic dinucleotide-and cyclic trinucleotide-responsive effectors 23 (Fig. 4a and Extended Data Fig. 9a, b).   To define the importance of degradation of cyclic nucleotide immune signals during phage infection, we infected E. coli expressing complete CBASS and Pycsar defence operons and quantified the effect of Acb1 and Apyc1 expression on phage replication. In the presence of an active type III CBASS operon from E. coli KTE188, Acb1 expression significantly boosted infectivity of the normally susceptible phage P1 by about 1.5 log (Fig. 4c). Likewise, expression of Apyc1 in E. coli disrupted Pycsar defence and completely rescued growth of phage T5, demonstrating that Acb1 and Apyc1 are sufficient to counteract host CBASS and Pycsar defence (Fig. 4d). To determine whether cyclic nucleotide degradation is necessary for immune evasion, we next focused on engineering a mutant phage lacking the ability to cleave immune nucleotide signals. Robust approaches do not yet exist for genetic manipulation of B. subtilis phages, and analysis of apyc1-deletion viruses will therefore be a focus of future research. However, we were able to use recent advances in coliphage engineering to create a phage T4 mutant virus lacking functional Acb1 (phage T4 Δacb1) (Extended Data Fig. 10a). E. coli cells infected with phage T4 Δacb1 do not hydrolyse 3′3′-cGAMP, confirming that Acb1 is essential for viral degradation of CBASS immune cyclic nucleotides (Extended Data Fig. 10b). In the absence of functional CBASS defence, phage T4 and phage T4 Δacb1 grow equally well, revealing that Acb1 is not required for normal replication in E. coli (Fig. 4e, f and Extended Data Fig. 10c, d). In contrast, growth of phage T4 Δacb1 is specifically impaired in the presence of active CBASS immunity with the mutant virus exhibiting a >300-fold defect in viral replication compared to wild-type phage T4 (Fig. 4e, f and Extended Data Fig. 10c, d). These results demonstrate that viral nucleases are critical for evasion of cyclic nucleotide-mediated phage defence.
Together, our data define Acb1 and Apyc1 as founding members of families of anti-CBASS and anti-Pycsar immune evasion proteins that allow phages to selectively hydrolyse cyclic nucleotide immune signals used for host defence. No single phage could degrade all cyclic nucleotide immune signals, revealing that diversification of cyclic nucleotide signals between CBASS and Pycsar systems is a key host adaptation to maintain successful antiphage defence 2,11 . Acb1 and Apyc1 join a growing collection of viral nuclease enzymes dedicated to immune evasion, including phage ring nucleases that degrade cA 4 and cA 6 signals used in type III CRISPR immunity 27,28 and poxin enzymes that degrade 2′3′-cGAMP to inhibit cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling in animals 29 . Each of these viral enzymes is structurally distinct, demonstrating at least four separate instances of prokaryotic and eukaryotic viral evolution to degrade host cyclic nucleotide immune signals. The broad specificity of Acb1 allows evasion of diverse CBASS operons with a single gene, and the ability of Acb1 to cleave cyclic trinucleotide species suggests that this enzyme may also enable evasion of type III CRISPR systems that use cAAA signals. Notably, Acb1 is unable to cleave the non-canonical 2′-5′ linkage in the CBASS signalling molecule 3′2′-cGAMP (ref. 30 ), mirroring the recent demonstration that 3′2′-cGAMP signalling in animals enables resistance

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to poxin enzymes 31 . The large diversity of >180 possible nucleotide signals proposed to exist in antiphage defence suggests that in addition to signal degradation, phages may encode Acb and Apyc proteins that target alternative components of CBASS or Pycsar immunity. Overall, our results define viral nucleases as a widespread mechanism of CBASS and Pycsar immune evasion and reveal the role of viral proteins in driving evolution of cyclic nucleotide-based immune defence systems.

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In general, phage infections were performed in MMB media at 37 °C for E. coli MG1655 and at 30 °C for B. subtilis and phages were propagated by picking a single phage plaque into a liquid culture grown to an optical density at 600 nm (OD 600 ) of 0.3 in MMB medium until culture collapse. The culture was then centrifuged for 10 min at 3,200g, and the supernatant was filtered through a 0.2-μm filter. The titre of the lysate was determined using the small-drop plaque assay method as described previously 34 .

Recombinant protein expression and purification
Acb1, Apyc1, cGAS/DncV-like nucleotidyltransferases (CD-NTase), cGAS-like receptors and effector proteins were purified from E. coli as previously described 11 After overnight expression, cell pellets were collected by centrifugation and then resuspended and lysed by sonication in 50 ml lysis buffer (20 mM HEPES-KOH pH 7.5, 400 mM NaCl, 10% glycerol, 30 mM imidazole, 1 mM TCEP). Lysate was clarified by centrifugation at 50,000g for 30 min, supernatant was poured over 8 ml Ni-NTA resin (Qiagen), resin was washed with 35 ml lysis buffer supplemented with 1 M NaCl, and protein was eluted with 10 ml lysis buffer supplemented with 300 mM imidazole. Samples were then dialysed overnight in dialysis tubing with a 14 kDa molecular weight cutoff (Ward's Science), and SUMO2 tag cleavage was carried out with recombinant human SENP2 protease as previously described 35 . Proteins used for crystallography were dialysed overnight at 4 °C in dialysis buffer (20 mM HEPES-KOH pH 7.5, 250 mM KCl, 1 mM TCEP), and then purified further by size-exclusion chromatography using a 16/600 Superdex 75 column (Cytiva), whereas proteins used for biochemical assays were dialysed in dialysis buffer supplemented with 10% glycerol. Purified proteins were concentrated to >15 mg ml −1 using 10-kDa MWCO centrifugal filter units (Millipore Sigma), aliquoted, flash frozen in liquid nitrogen and stored at −80 °C.

Thin-layer chromatography
Thin-layer chromatography was used to analyse cyclic nucleotide degradation as previously described 29 . Cyclic nucleotides were synthesized using the following purified recombinant enzymes: V. cholerae DncV (ref. 11  Plates were dried at room temperature, exposed to a storage phosphor screen, and detected with a Typhoon Trio Variable Mode Imager System (GE Healthcare).  Table 1). Samples of 5 ml in volume were taken and centrifuged for 5 min at 3,200g and 4 °C. The culture pellets were flash frozen using dry ice and ethanol. E. coli pellets were resuspended in 250 μl of a lysis buffer containing 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM MnCl 2 , 1 mM DTT, 10% glycerol and 1% NP-40, and incubated at room temperature for 30 min with occasional vortexing. Bacillus pellets were first treated with T4 lysozyme (Ther-moFisher) at 1 mg ml −1 in PBS at 37 °C for 10 min, followed by addition of 400 μl of E. coli lysis buffer and 30-min incubation at room temperature. Samples were clarified by centrifugation for 5 min at 17,000g at 4 °C, and the supernatant was aliquoted and flash frozen in liquid nitrogen, and stored at −80 °C.

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and further separated with a 10/300 Superdex 75 column (Cytiva). In a separate approach, (NH 4 ) 2 SO 4 was added to clarified lysates to a final concentration of 30%, and precipitated proteins were removed by centrifugation at 20,000g for 15 min. The soluble fraction was then separated using hydrophobic interaction chromatography using a 5-ml phenyl column (Cytiva) and a gradient of 1-0.0 M (NH 4 ) 2 SO 4 . Active fractions were pooled, concentrated, and further separated with a 10/300 Superdex 200 column (Cytiva). For each enrichment scheme, phage T4 proteins enriched in fractions with the highest activity relative to neighbouring inactive fractions were quantified by label-free mass spectrometry as previously described 29 .
Phage T4 genes identified by biochemical fractionation and mass spectrometry were amplified from genomic T4 DNA isolated from infected E. coli using a Qiagen DNeasy Blood and Tissue kit as described previously 37 . Candidate genes were PCR amplified using Q5 DNA polymerase (NEB) and primers designed to incorporate a 49-base-pair sequence containing a T7 promoter and a ribosome-binding site upstream of the amplified candidate gene according to the NEB cell-free E. coli protein synthesis system instructions (NEB). PCR products were purified using a PCR clean-up kit (Qiagen) and translated using the E. coli protein synthesis system kit (NEB). A 1 μl volume of each translation reaction was used to test for 3′3′-cGAMP cleavage activity by thin-layer chromatography. Acb1 was identified as the product of the phage T4 gene 57B.
Phage genome sequencing, assembly and annotation of SBSphiJ1-7 SBSphiJ1-7 phages were isolated from soil samples on B. subtilis BEST7003 culture as described previously 33 . High-titre phage lysates (>10 7 PFUs ml −1 ) were used for DNA extraction. A 500 μl volume of the phage lysate was treated with DNase-I (Merck catalogue number 11284932001) added to a final concentration of 20 mg ml −1 and incubated at 37 °C for 1 h to remove bacterial DNA. DNA was extracted using the QIAGEN DNeasy blood and tissue kit (catalogue number 69504) starting from the Proteinase-K treatment step to lyse the phages. Libraries were prepared for Illumina sequencing using a modified Nextera protocol as previously described 38 . Following Illumina sequencing, adapter sequences were removed from the reads using Cutadapt version 2.8 (ref. 39 ) with the option -q 5. The trimmed reads from each phage genome were assembled into scaffolds using SPAdes genome assembler version 3.14.0 (ref. 40 ), using the --careful flag. Each assembled genome was analysed with Prodigal version 2.6.3 (ref. 41 ; default parameters) to predict open reading frames.

SBSphiJ Apyc1 bioinformatic identification
The genomic sequences of SBSphiJ and the closely related family members SBSphiJ1-7 were aligned using progressive Mauve (ref. 42 ). Regions that were exclusive to cCMP-cleaving phages revealed eight candidate genes. The corresponding SBSphiJ protein sequences were analysed using HHpred (ref. 43 ) for predicted structural homologues. Protein classes with >75% probability are listed in Extended Data Fig. 5b and Apyc1 was identified as the product of the phage SBSphiJ gene 147.

Identification of Acb1 and Apyc1 homologues and generation of phylogenetic trees
Homologues of Acb1 and Apyc1 were identified using NCBI BLASTp with default parameters. Acb1 sequences were classified as belonging to a prophage if they were within three genes of a phage structural or packaging protein. Apyc1 phage sequences were identified by restricting the search to only viral sequences (NCBI taxid: 10293; https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax. cgi?id=10239). Maximum-likelihood trees were generated using the IQ-TREE web server with ultrafast bootstrapping and 1,000 iterations 44 . Consensus trees were then edited visually using the Interactive Tree Of Life 45 .

HPLC
Acb1 and Apyc1 reactions for HPLC analysis were performed in a 100 μl volume and consisted of 50 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM DTT, 100 μM chemically synthesized nucleotide standards (Biolog Life Science Institute) and 1 μM recombinant protein unless otherwise indicated. Apyc1 reactions were further supplemented with 5 mM MgCl 2 and 1 mM MnCl 2 . Reactions were incubated at 37 °C for 20 min (unless otherwise indicated in the figure legend) and filtered using a 10-kDa cutoff filter (Millipore). Filtered nucleotide products were analysed using a C18 column (Agilent Zorbax Bonus-RP 4.6 × 150 mm, 3.5 μm) heated to 40 °C and run at 1 ml min −1 in a buffer of 50 mM NaH 2 PO 4 adjusted to pH 6.8 with NaOH, supplemented with 3% acetonitrile.

Phage challenge assays
Phage challenge experiments were performed as previously described 1,2 by spotting serial dilutions of high-titre phage stocks onto a lawn of bacteria carrying a complete CBASS or Pycsar defence operon. The following defence systems were used: E. coli strain KTE188 (IMG gene accession numbers: 2564596481-2564596485; https://img.jgi.doe. gov/) cloned under its native promoter into the plasmid pSG1 (ref. 3 ), E. coli CdnG cloned under its native promoter into the plasmid pLOCO2 (ref. 23 ), Y. aleksiciae CdnE (ref. 36 ) cloned into a pBAD vector, and E. coli PycC (ref. 2 ) cloned under its native promoter into the plasmid pSG1. For EcCdnG and YaCdnE operons, control plasmids were also used in which the CD-NTase is inactivated (CdnG-D82A/D84A) 23 or the transmembrane segment of the receptor is deleted (YaCdnE) 36 . Phage replication in the context of these defence systems was measured using a spot plaque assay 36 . Briefly, E. coli MG1655 (EcKTE188, EcPycC) or BL21 cells (EcCdnG and YaCdnE) containing the defence systems were grown overnight at 37 °C. A 300 μl volume of the bacterial culture was mixed with 4 ml melted MMB agar containing appropriate antibiotics and 0.2% arabinose for pBAD plasmids, poured on top of a 15-cm plate of lysogeny broth and left to solidify in a plate for 1 h at room temperature. High-titre phage stocks were serially diluted tenfold in MMB and 3-5-μl drops were placed on the bacterial layer and allowed to dry at room temperature for 1 h. Plates were incubated overnight at 37 °C (Acb1 and Apyc1 rescue experiments) or 30 °C (Δacb1 T4 phage challenges) and plaque-forming units (PFUs) were determined by counting the derived plaques after overnight incubation. Phage infection of cells expressing active CBASS operons did not generate clear plaques. For these, the dilution at which there was no detectable defect in bacterial growth was counted as having a single plaque. For in vivo rescue experiments, acb1 and apyc1 were amplified from the genome of T4 phage or SBSphiJ phage and cloned into the plasmid pBbS8k (Addgene number 35276) using Gibson assembly (NEB).

Generation of phage T4 ΔAcb1
Nonsense mutations were introduced into acb1 using a CRISPR-based selection strategy as described previously 52,53 . Briefly, a gRNA targeting acb1 and a repair template with nonsense mutations were cloned into pCRISPR (Addgene 42875). E. coli Top10 cells were then transformed with the pCRISPR-gRNA-acb1 repair plasmid and pCas9 (Addgene 42876). A colony was picked, and 2-ml log-scale cultures were infected with WT phage T4 until culture collapse. The resulting lysate was filtered through a 0.22-μM filter and plated on E. coli Top10 cells with no plasmid. Single plaques were picked into 200 μl SM buffer (50 mM Tris-HCl pH 8.5, 100 mM NaCl, 8 mM MgSO 4 ) containing 2 μl chloroform. After 1-h incubation at room temperature, 4 μl was used as input for standard PCR reactions using GoTaqGreen (Promega) according to the manufacturer's instructions. PCR products were purified using QIAquick gel extraction kit (Qiagen) and sequenced for introduction of nonsense mutations. Positive phage T4 clones went through three rounds of plaque purification before generating a high-titre stock used in all phage challenge experiments.

Statistics and reproducibility
Statistical tests are described in the figure legends and were performed using GraphPad Prism 9.3.1. Experimental details regarding replicates and sample size are described in the figure legends. No statistical methods were used to predetermine sample size and no blinding or randomization was used for this study.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
Coordinates and structure factors of FBB1 Acb1, the FBB1 Acb1-3′3′-cGAMP complex, Bsp38 Apyc1, P. J14 Apyc1 and P. xerothermodurans Apyc1 have been deposited in the PDB under the accession codes 7T26, 7T27, 7T28, 7U2R and 7U2S, respectively. Source data are provided with this paper. All other data are available in the manuscript or the Supplementary Information.          Corresponding author(s): Philip Kranzusch Last updated by author(s): Mar 24, 2022 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
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Software and code
Policy information about availability of computer code Data collection Protein homologs were identified using NCBI BLAST.
Phylogenetic trees were constructed using the IQ-TREE web server v1.6.12. Radiographic images were collected using Typhoon scanner control 2.0.0.6 Chromatography traces were collected using GE Unicorn 7.1 DNA gel images were collected using BioRad Quantity One 4.6.9 Data analysis Phenix 1.19, Coot 0.8.9, PyMOL 2.3, Prism 9.3.1, iTOL v6 For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Coordinates and structure factors of FBB1 Acb1, the FBB1 Acb1-3'3'-cGAMP complex, Bsp38 Apyc1, Paenibacillus J14 Apyc1, and Paenibacillus xerothermodurans Apyc1 have been deposited in PDB under the accession codes 7T26, 7T27, 7T28, 7U2R, and 7U2S, respectively. All other data are available in the manuscript or the supplementary materials.